1. Field of the Invention
The present invention generally concerns a new method of engineering a kidney in vitro.
The present invention particularly concerns a new method and procedure for propagating cloned kidney members from embryonic ureteric bud tips grown in vitro under specific culture conditions.
2. Description of Related Art
Branching tubulogenesis is an essential mechanism by which epithelial tissues such as kidney, salivary gland and prostate develop (Proc. Natl. Acad. Sci. USA, 96, 7330–7335, 1999 incorporated herein by reference). Largely based on the classical studies of Brobstein and coworkers, direct interactions between mesenchymal and epithelial components of embryonic tissue have been thought to be crucial for branching morphogenesis in most epithelial tissues. During kidney development, for example, direct cell-cell interactions between the metanephric mesenchyme and the epithelial component, the ureteric bud (UB), are believed to be essential for branching morphogenesis of the latter. This view is based on the fact that it had not been possible, in many previous studies, to observe proliferation and branching of the UB in the absence of direct contact with the metanephric mesenchyme or another inducing tissue, suggesting that the developmental program necessary for branching depended upon direct contact between surface proteins of the UB with surface proteins of the metanephric mesenchyme. Further, no known soluble factor or set of factors had been able to induce UB branching morphogenesis in vitro.
This view has gained additional support from knockout experiments in which absent expression of a variety of individual soluble growth factors held to be important in kidney development, based upon previous organ culture experiments, fail to show defective branching morphogenesis of the UB. Nevertheless, recent studies have also shown that glial cell line derived neurotrophic factor (GDNF) is necessary for early UB outgrowth, but on its own, it fails to promote proliferation and branching morphogenesis of isolated UB in vitro. These results left open the possibility that some unknown soluble factor, or combination of factors, derived form the metanephric mesenchyme, might be sufficient to induce epithelial branching morphogenesis.
A wide array of renal and urological abnormalities are likely due to defective tubulogenesis and branching morphogenesis of the developing collecting system
It is now clear that a variety of defects in the kidney and urinary collecting system are the result of abnormal development of these structures in the fetus. The spectrum of disease is huge, as is their potential morbidity. The molecular basis for these diseases is poorly understood; however, it appears, in many instances, that the problem lies in defective morphogenesis of the ureteric bud. The urinary collecting system (from the trigone of the bladder including ureteral orifices, ureters, renal pelvis, and collecting tubules) arises from the UB. Thus, developmental abnormalities of the UB and its derivatives would be expected to give rise to a variety of “urological” as well as “nephrological” clinical syndromes. Developmental anomalies extrinsic to the kidney, but in principle attributable to defects in UB morphogenesis, include vesicoureteral reflux (VUR), ureteropelvic junction obstruction (UPJO), ectopic and duplicated ureters. Since normal kidney development depends critically upon mutual inductive interactions between the UB and the metanephric mesenchyme (MM), inefficient or defective branching morphogenesis of the UB would be expected to result in various aplastic, hypoplastic, and perhaps dysplastic diseases. Extrinsic collecting system abnormalities would then be expected to, coexist with various hypoplastic diseases of the kidney. In fact, up to ⅓ of end stage renal disease in children is due to developmental problems, the majority of which may be categorized as ureteral with or with out dysplasia or hypoplasia of one or both kidneys. In addition, perhaps 5–10% of all adults has some occult developmental anomaly.
Recent advances in the molecular biology of kidney development demonstrate that specific molecular defects can explain a variety of clinical syndromes. VUR, UPJO, and various dysplastic, and hypoplastic kidney disorders have been known to co-exist and to be expressed in various human lineages with a variable penetrance, the so-called CAKUT syndrome. In addition, several targeted gene-deletion experiments have resulted in phenotypes that may be best characterized as resulting form defective UB morphogenesis (directly or indirectly). These range from the renal aplasia associated with complete UB failure associated with deletions of WT-1 and RTK c-ret molecules to more subtle effects resulting in hypoplasia or oligonephronia such as seen with certain integrin knockouts. Defective collecting system development may play a role in the most common congenital cystic disease, ADPKD. The inventors have shown that expression of PKD-1 correlates spatiotemporally with branching morphogenesis of the UB. Findings such as this have led to the hypothesis that ADPKD and other cystic diseases of the kidney result form defects in the developmental program necessary for proper tubulogenesis.
Aside from “congenital” disease per se, defective collecting system development may underlie predisposition to disease much later in life. It has been argued that low nephron number is crucial to the development of hypertension and chronic renal failure in adults. This may well be the result of defective branching morphogenesis during development of the urinary collecting system, because the degree of ureteric bud branching during collecting system development determines the number of nephrons in the adult kidney. Hence, aggregate nephron number is a function of factors regulating ureteric bud branching during urinary tract development. If one assumes a 1% decrement in efficiency of branching morphogenesis (99% efficient at all steps), this results in less than half the normal number of nephrons after the roughly 20 generations of branching which occur during human nephrogenesis.
In summary, a broad spectrum of disorders ranging from urological abnormalities, hypoplasia, dysplasia, and cystic diseases, and possibly even certain forms of “essential” hypertension, may be viewed as developmental diseases of ureteric bud and its derivatives. Recent work indicates that a molecular basis exists for these disorders and that much human morbidity and mortality may be attributable to varying degrees of failure in the process of ureteric bud branching morphogenesis.
The cellular and molecular basis of development of the urinary collecting system, particularly tubulogenesis and branching morphogenesis, are not well understood
In the mouse, inductive interactions between the MM and the UB that are necessary for formation of the metanephric kidney take place around embryonic day 11; in the rat, this occurs around day 13. Through in vitro organ and cell culture studies, as well as knockouts, both soluble factor influence and cell—cell contact have been implicated, although the exact nature of the inducing signals is a topic of intense investigation and debate. Subsequent to these interactions, the metanephric mesenchyme undergoes a “mesenchymal to epithelial transition,” during which it acquires epithelial markers such as cytokeratins. As development progresses, the recently epithelialized mesenchyme forms early nephronal structures, which ultimately develop into the proximal through distal tubule. All this appears to be guided by interactions with the ureteric bud as it, through a process of branching morphogenesis, develops into the collecting system. Thus, while the mesenchyme is differentiating, the ureteric bud is invading it and undergoing iterations of symmetric and asymmetric dichotomous branching. About 20 generations of such branching events result in the roughly 1 million collecting ducts that form the renal portion of the urinary collecting system.
This general process is not unique to the kidney. Branching tubulogenesis (ductogenesis) is an essential mechanism by which most, if not all, epithelial tissues form in the embryo. Largely due to the classical studies using organ culture, direct interactions between mesenchymal and epithelial components of embryonic tissue have been thought to be crucial for branching morphogenesis in kidney and urinary tract. This view is based on the fact that it had not been possible, in many previous studies, to observe proliferation and branching of the UB in the absence of direct contact with the metanephric mesenchyme or another inducing tissue, suggesting that the developmental program necessary for branching depended upon direct contact between surface proteins of the UB with surface proteins of the metanephric mesenchyme. Furthermore, no known soluble factor or set of factors had been able to induce UB branching morphogenesis in vitro. This view has gained additional support from knockout experiments in which absent expression of a variety of individual soluble growth factors held to be important in kidney development (based upon previous organ culture experiments) fail to show defective branching morphogenesis of the UB. Nevertheless, recent studies have demonstrated that glial cell line derived neurotrophic factor (GDNF) is necessary for early UB outgrowth, but as the inventors have shown, on its own, it fails to promote proliferation and branching morphogenesis of isolated UB in vitro (FIG. 6). [Proc. Natl. Acad. Sci., 96, 7330–7335, 1999 incorporated herein by reference].
The primary focus of this invention is to present a novel method and procedure for propagation of cloned kidney members from embryonic ureteric bud which also has applicability to other epithelial-derived tissues. This method differs in concept and substance from U.S. Pat. No. 6,060,270 (May 9, 2000) issued to Humes. In contrast to the Humes patent, this method employs the intrinsic ability of the embryonic epithelial tissue to branch in order to generate an indefinite number of organs from a single embryonic ureteric bud. Thus, in principle, after six generations of branching, a single ureteric bud can give rise to 256 (28) kidneys or even more, depending upon the number of generations the ureteric bud is allowed to branch in culture.